JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY
VOL. 68, NO. 23, 2016
ª 2016 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION
ISSN 0735-1097/$36.00
PUBLISHED BY ELSEVIER
http://dx.doi.org/10.1016/j.jacc.2016.09.945
REVIEW TOPIC OF THE WEEK
MicroRNAs in Cardiovascular Disease
Temo Barwari, MD, Abhishek Joshi, BA, BMBCH, Manuel Mayr, MD, PHD
ABSTRACT
Micro-ribonucleic acids (miRNAs) are in the spotlight as post-transcriptional regulators of gene expression. More than
1,000 miRNAs are encoded in the human genome. In this review, we provide an introduction to miRNA biology and
research methodology, and highlight advances in cardiovascular research to date. This includes the potential of miRNAs
as therapeutic targets in cardiac and vascular disease, and their use as novel biomarkers. Although some miRNA therapies
are already undergoing clinical evaluation, we stress the importance of integrating current knowledge of miRNA biology
into a systemic context. Discovery studies focus on miRNA effects within one specific organ, whereas the expression
of most miRNAs is not restricted to a single tissue. Because most miRNA-based therapies act systemically, this may
preclude widespread clinical use. The development of more targeted interventions will bolster well-informed clinical
applications, increasing the chances of success and minimizing the risk of setbacks for miRNA-based therapeutics.
(J Am Coll Cardiol 2016;68:2577–84) © 2016 by the American College of Cardiology Foundation.
O
nly 1% of the human genome codes for genes
region, a sequence of 6 to 8 nucleotides that binds to
that function in protein synthesis (1). The
messenger ribonucleic acid (mRNA), the so-called
remaining 99% of deoxyribonucleic acid
miRNA targets (3). MiRNA synthesis and silencing
(DNA) was initially considered to be junk. It is now
have both been extensively reviewed recently (4,5).
recognized that the majority of the genome may have
The key biological concepts are summarized in the
biochemical functions, representing regulatory, non-
Central Illustration. Initially, a precursor transcript is
coding ribonucleic acid (RNA). Several subcategories
produced and then forms double-stranded RNA.
of noncoding RNAs exist, in particular, long noncoding
Later, the miRNA duplex undergoes unwinding,
RNAs and small noncoding RNAs. Among the latter,
whereby only a single strand, the so-called guide
microRNAs (miRNAs/miRs) have thus far attracted
strand, which is usually the functional unit, is loaded
most attention since their discovery in Caenorhabditis
in the RNA-induced silencing complex (RISC). The
elegans (2). MiRNAs affect the production of proteins
other strand or passenger strand is often degraded,
by interacting with transcribed messenger RNAs
but may also function as a mature miRNA (6). In the
(mRNAs), thus silencing the expression of genes.
RISC, the miRNA binds to its target mRNA, prevent-
Here, we aim to provide an overview of miRNA biology
ing its translation into a protein. Single miRNAs
for
suppress more than 1 gene, and miRNAs with similar
clinicians,
discussing
their
therapeutic
and
diagnostic potential, as well as their limitations.
seed regions may suppress a similar, but nonidentical, set of genes, and to differing degrees. Gene
BASIC BIOLOGY OF miRNAs
suppression is usually partial, rather than total, and a
single gene can have binding sites for multiple
MiRNAs are short (w22 nucleotides), noncoding RNA
miRNAs. This organizational complexity, illustrated
molecules. They exert their function via the seed
by a high false-positive rate of target prediction
Listen to this manuscript’s
audio summary by
JACC Editor-in-Chief
From the King’s British Heart Foundation Centre, King’s College London, London, United Kingdom. Dr. Barwari is an Interdis-
Dr. Valentin Fuster.
ciplinary PhD student funded by the British Heart Foundation (BHF). Dr. Joshi has been awarded a BHF Clinical Research Training
Fellowship. Dr. Mayr is a BHF Senior Research Fellow (FS/13/2/29892) and supported by the Fondation Leducq (MIRVAD; 13 CVD 02)
and the NIHR Biomedical Research Center based at Guy’s and St. Thomas’ National Health Service Foundation Trust and King’s
College London, in partnership with King’s College Hospital. King’s College London and Prof. Mayr hold patents on microRNA
biomarkers. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
Manuscript received July 18, 2016; revised manuscript received September 12, 2016, accepted September 13, 2016.
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MicroRNAs in Cardiovascular Disease
DECEMBER 13, 2016:2577–84
ABBREVIATIONS
algorithms (7), presents challenges in both
mechanism
AND ACRONYMS
understanding the functions of miRNAs and
in most cases, overexpression of a miRNA will
manipulating their effects.
suppress its direct targets, whereas inhibiting an
anti-miRs = inhibitors of
for
manipulating
protein
synthesis;
endogenous miRNA will de-repress their expression.
miRNAs
CVD = cardiovascular disease
ECM = extracellular matrix
MEASUREMENT OF miRNAs
Unmodified RNA strands are degraded upon
administration; thus, miRNA therapeutics require
miRNAs are relatively stable, and can be
either efficient methods of cell type-specific delivery
reliably measured in tissues, as well as in
or modifications that enhance stability but preserve
MI = myocardial infarction
biofluids (8). Several techniques have been
miRNA function. For now, clinical studies with
miRNA/miR = micro-
developed to identify and quantify miRNAs.
miRNA
Benefits
different
miRNAs (anti-miRs). Anti-miRs are synthetic single
techniques have been summarized previ-
strands of RNA, consisting of complementary nucle-
ously elsewhere (8). Here, we briefly discuss
otides to an endogenous miRNA. Various structural
MHC = myosin heavy chain
ribonucleic acid
mRNA = messenger ribonucleic
acid
RISC = ribonucleic acidinduced silencing complex
SMC = smooth muscle cell
and
disadvantages
of
the most commonly used methods.
therapeutics
mainly
use
inhibitors
of
modifications have been designed to increase their
Real-time quantitative polymerase chain
half-life in the circulation, bypass degradation in
reaction has been the cornerstone for miRNA
tissues and enhance intracellular delivery (10). Car-
quantification and remains the most reliable tech-
diotropic adeno-associated viruses achieve efficient
nique for quantitative comparison of miRNA expres-
cardiomyocyte-specific miRNA delivery (11). The
sion levels. This technique uses predefined primers to
translational potential of adeno-associated virus–
amplify and measure individual miRNAs in a sample.
mediated oligonucleotide delivery has been reviewed
Microarrays use hybridization of miRNAs to specific
elsewhere (12). Currently, overexpressing a miRNA is
primers, trading less accurate quantification for
generally considered less safe than inhibiting an
higher throughput and lower cost, and measuring
endogenous miRNA.
hundreds of miRNAs in parallel. Because both of
Miravirsen is an anti-miR targeting miR-122 for
these techniques rely on predefined primer se-
treatment of hepatitis C (13), which has completed a
quences, they are not able to discover previously
multicenter phase 2a trial (14) and is currently in a
uncharacterized miRNAs.
phase 2b trial. The choice of miR-122 as the first
RNA sequencing techniques provide “hypothesis-
therapeutic target highlights the challenges when
free” identification of RNA species, allowing the
targeting cardiovascular disease (CVD). First, miR-
discovery of new miRNAs and quantitative analysis
122 shows exquisite tissue specificity, whereas most
of a comprehensive miRNA transcriptome. The
miRNAs identified as treatment targets for CVD are
use of computational solutions to resolve reads
ubiquitously expressed, raising concerns for off-
into miRNAs suffers from the risk of reporting
target effects. Second, anti-miRs predominantly
putative sequences that do not have real-world
accumulate in the liver and kidneys, circumventing
correlates (9).
Without added spike-ins and standard curves, all
the need for tissue-specific targeting (13). The latter
is further illustrated by the evaluation of anti-miR-21
techniques rely on relative rather than absolute
as a therapy for Alport nephropathy (15). These
quantification, meaning that differences in miRNAs
ongoing clinical trials will provide more insight into
are presented as a “fold change” between paired
the practical use of miRNA therapeutics.
samples, and not as an absolute unit, requiring in-
Targeting the heart or vasculature with systemic
formation on the context of abundance. Experimental
anti-miRs would require significantly higher dosing,
work must show downstream effects of miRNA
and efficiency may be low. Animal models have shown
changes as readout for miRNA function, specifically
nephrotoxicity at higher doses of some anti-miRs,
by comparing the profiles of multiple miRNAs with
although the clinical trial of Miravirsen did not find
differential expression of target proteins. Ideally, the
evidence for renal injury in humans (14). The human
miRNA/mRNA duplexes in the RISC are analyzed to
immune system has evolved to detect viral RNA.
prove direct interactions.
Toll-like receptors recognize both single- and double-
THERAPEUTIC MANIPULATION OF miRNAs
cleotides may elicit an immune response that could
The central action of miRNAs is to suppress protein
applications will require solutions for local or cell-
stranded RNA (16). High doses of synthetic oligonucompromise efficacy and safety. Thus, cardiovascular
expression through binding and silencing specific
type–specific delivery, and clinically detectable,
target mRNAs, which, in turn, reduces protein
reliable
synthesis. Therefore, miRNAs offer a tantalizing
engagement.
readouts
to
monitor
successful
target
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MicroRNAs in Cardiovascular Disease
miRNAs IN HEART FAILURE
failure were announced in 2011, but have not
progressed further.
At the cellular level, heart failure is caused by car-
In 1 study, miR-25 expression was repressed in
diomyocyte dysfunction and fibrosis due to accumu-
failing human hearts (29), but its expression was
lation of extracellular matrix (ECM). These processes
increased in another study (30). Where the former
remain almost entirely untreated by standard heart
study described the targeting of deleterious embry-
failure treatment regimens.
onic gene programs that worsen cardiac function, the
miR-133 is highly abundant in cardiomyocytes, but
latter showed repression of the sarcoplasmic/endo-
is reduced in animal models of hypertrophy and in
plasmic reticulum calcium adenosine triphosphatase
patients with hypertrophic cardiomyopathy (17).
(SERCA), an important contributor to excitation–
In vitro and in vivo studies showed increased hyper-
contraction coupling in cardiomyocytes, and subse-
trophy upon miR-133 inhibition, and preserved car-
quent improvement in cardiac function. Differences in
diac
overexpression.
timing and chemical properties of the anti-miR treat-
Targeting of the beta-1 adrenergic receptor pathway,
ment, as well as the study duration, could explain
central to the progression and treatment of heart
these contradictory results. This highlights the formi-
failure, was implicated as the underlying mechanism
dable task of determining optimal anti-miR chemistry,
function
upon
miR-133
(18). In addition to the heart, miR-133 is also present
given the combinatorial possibilities of modifications
in skeletal muscle, albeit at lower levels than in car-
that can be introduced, even in small oligonucleotides.
diomyocytes. Here, miR-133 inhibition again in-
Different oligonucleotides targeting the same miRNA
creases responsiveness to adrenergic stimuli and
may not achieve the same therapeutic benefit.
promotes differentiation to brown fat tissue (19).
miR-133 manipulation also seems to affect the cardiac
action potential (20).
MiR-1 is part of the same cluster as miR-133 and
shares its abundance, as well as its lower expression
in heart failure patients (21). Both increased (22) and
reduced (23) expression of miR-1 lead to electrophysiological abnormalities. Interestingly, miR-1 targets
insulin-like
growth
factor-1,
which
itself
represses processing of the pre-miR-1 transcript (21).
The insulin-like growth factor-1 pathway is an
important contributor to cardiac hypertrophy and
arrhythmias, and increasing miR-1 levels seems to
improve cardiac function (24).
MiR-208 is also highly enriched in cardiomyocytes,
and regulates the balance between the a - and
b-myosin heavy chains (MHC). Induction of the
b-MHC isotype is a known maladaptive response to
cardiac stress and reduces contractility (25). MiR-208
knockout mice, and rats treated systemically with
anti-miR-208, had preserved balance between both
MHC isotypes in response to experimental cardiac
stress, with better cardiac function (26). MiR-208 inhibition has therefore been suggested as a protective
treatment in heart failure. However, b-MHC expres-
miRNAs IN CARDIAC REGENERATION
MiRNAs have been proposed as an alternative to cell
therapy for cardiac regeneration. Studies on neonatal
rat cardiomyocyte proliferation highlighted miR-199a
and miR-590 as capable of inducing mitosis (31).
Injecting these miRNAs into rodent hearts after
myocardial infarction (MI) preserved cardiac function. Along similar lines, inhibition of miR-34a
improved cardiac function after MI in mice, attenuating cardiomyocyte apoptosis and telomere shortening
(32).
MiRNA-based
therapies
for
cardiac
regeneration and repair still require validation in
models with greater translational potential. A miRNA
that is pursued currently for therapeutic applications
in CVD is miR-92a. Inhibition of miR-92a reduces
endothelial inflammation (33) and promotes angiogenesis and functional recovery in ischemic myocardium (34). However, this miRNA is part of a cluster of
miRNA genes (miR-17w92), also known as oncomiR-1
because its members target cell-cycle regulation. This
raises concerns about potential side effects of miR92a therapeutics.
miRNAs IN CARDIAC FIBROSIS
sion is not altered in normal hearts of miR-208
knockout mice, indicating that effects of this miRNA
Several miRNAs have been implicated in cardiac
are either dependent on disease context or subject to
fibroblast survival and related signaling pathways.
parallel controls (27). Furthermore, recent deep
Although some miRNAs directly target genes coding
sequencing data from human hearts suggest that miR-
for ECM proteins (35,36), others prevent cardiac
208 expression is relatively low compared with other
fibroblasts from attaining an activated secretory
cardiac miRNAs, such as miR-1 and miR-133 (28). Pre-
phenotype (37,38). In addition to its role in cardiac
clinical trials using miR-208 inhibition in heart
hypertrophy,
miR-133
is
considered
antifibrotic
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DECEMBER 13, 2016:2577–84
C E NT R AL IL L U STR AT IO N miRNA Biogenesis and Function
Barwari, T. et al. J Am Coll Cardiol. 2016;68(23):2577–84.
Continued on the next page
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MicroRNAs in Cardiovascular Disease
through targeting of connective tissue growth factor,
miR-29b antagonism could more subtly alter the ECM
a key regulator of the fibrotic process (37), as well as
balance, if stents eluting anti–miR-29b were devel-
collagen I a -1, a main constituent of the cardiac ECM
oped to inhibit aneurysm progression or to stabilize
(39).
symptomatic atherosclerotic plaques.
MiR-21 has been studied most extensively in the
Key events in atherosclerosis are endothelial injury
context of fibrosis. This miRNA is increased in heart
and the switch of SMCs from a contractile to a syn-
failure patients and in cardiac fibroblasts of fibrotic
thetic phenotype. MiR-143 and miR-145 are tran-
mouse hearts (40), and promotes ECM deposition in
scribed together as a cluster and are highly abundant
mouse models of increased afterload (41) and
in SMCs, with a marked down-regulation seen in
myocardial ischemia (42). In vivo inhibition of miR-21
vessels with neointima formation (50,51). Together,
attenuates the fibrotic response and improves cardiac
these miRNAs regulate vascular SMC differentiation
function in mouse models of heart failure (41). These
and, consequently, their loss contributes to SMC
results were not reproduced in a subsequent study
dedifferentiation and atherosclerosis (52). MiR-126
using a different anti-miR, reiterating the importance
is highly enriched in endothelial cells (53). It indi-
of optimizing the anti-miR chemistry (43). MiR-21 also
rectly enhances vascular endothelial growth factor
serves as an example of a miRNA where both the
signaling, and therefore has been studied in the
guide and passenger strands mediate function,
context of angiogenesis and endothelial repair.
with the passenger strand being transferred from
Interestingly, the endothelial effects of this miRNA
fibroblasts to cardiomyocytes, where it exerts a pro-
seem to be mediated by the passenger strand, rather
hypertrophic effect (6). This highlights another diffi-
than the guide strand (54). Although miR-126 has
culty in translating findings from preclinical models
been described as endothelial cell-specific (53), this
to patients. If both strands mediate function, then
miRNA is expressed in megakaryocytes and may have
inhibition of just 1 strand by anti-miR treatment may
a role in platelet function as mentioned elsewhere in
not recapitulate the phenotype observed in knockout
this paper.
mice, where both strands are deleted from the
genome.
miRNAs IN LIPID METABOLISM
miRNAs IN NEOINTIMA FORMATION
MiR-122 is highly abundant in the liver (55,56). Phar-
AND ATHEROSCLEROSIS
macological
strategies
to
lower
miR-122
levels
decreased plasma cholesterol levels (13,55). UnfortuIn addition to its profibrotic role, miR-21 enhances
nately, initial optimism was dampened by subse-
neointimal growth through pro-proliferative and
quent studies that showed a simultaneous decrease in
antiapoptotic effects on vascular smooth muscle cells
high-density
(SMCs) (44). Inhibition of miR-21 reduces in-stent
Similar findings were obtained for miR-33, an miRNA
restenosis in animals (45). The development of
that regulates cholesterol metabolism. Short-term
lipoprotein
cholesterol
levels
(57).
miRNA-eluting stents (46) could overcome one of the
inhibition was beneficial (58,59), but long-term inhi-
major challenges in miRNA therapies, because local
bition in animals fed a high-fat diet had detrimental
delivery decreases the risk of off-target effects. The
effects, such as hepatic steatosis (60). More recently,
same can be argued for miR-29b, which represses
a study in human hepatic cells identified miR-148a as
ECM production by vascular SMCs (47), whereas in-
a regulator of the low-density lipoprotein cholesterol
hibition slows abdominal aortic aneurysm progres-
receptor (61). Systemic inhibition of miR-148a caused
sion (48) and promotes favorable plaque remodeling
a significant reduction in plasma low-density lipo-
in
protein cholesterol, but also increased high-density
atherosclerotic
mice
(49).
Delivered
locally,
C ENTR AL I LL U STRA T I O N Continued
MicroRNAs (miRNAs) originate from primary transcripts (pri-miRNAs) that are derived from introns (the noncoding regions within a primary mRNA transcript) of
protein-coding genes or from intergenic regions within the genome. Primary transcripts are processed in the nucleus to a hairpin-shaped pre-miRNA by the Drosha/
DGCR8 complex, transported to the cytoplasm, and then processed to mature miRNA duplexes by the Dicer complex. To exert its function, the mature miRNA is
incorporated into an RNA-induced silencing complex (RISC). This complex can then target mRNA through sequence complementarity: the sequence of the incorporated
miRNA, with the 6 to 8 nucleotide-long seed sequence on the 50 end in particular, binds to the targeted mRNA, usually to the untranslated region at the 30 end.
Depending on several factors, including the extent of sequence complementarity, this leads to cleavage or translation repression of the mRNA, preventing a protein from
being assembled. mRNA ¼ messenger RNA; RISC ¼ ribonucleic acid-induced silencing complex; tRNA ¼ transfer RNA.
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DECEMBER 13, 2016:2577–84
lipoprotein cholesterol levels. Long-term side effects
between 2 of these miRNAs and platelet function:
of miR-122 and miR-33 inhibition, combined with the
miR-126 (75) and miR-223 (76). Circulating miRNAs
advent of novel therapeutic options for dyslipidemia,
can be derived from a range of cell types, but plate-
may limit the clinical use of miRNAs to modulate lipid
lets contribute substantially (75,76). Levels of circu-
metabolism.
lating miRNAs are affected by the administration of
antiplatelet therapy (77), and correlate with existing
miRNAs AS BIOMARKERS
platelet reactivity assays in patients post-MI (75).
MiRNAs are present, stable, and detectable in the
circulation (62), both in plasma or serum, where they
are either bound to protein complexes or contained in
microvesicles
or
lipoproteins.
Microvesicle-
Platelet miRNAs have been linked to hyper-reactive
platelets (78).
CONCLUSIONS
and
lipoprotein-borne miRNAs have been suggested to
MiRNAs can regulate protein expression, and so are
affect protein expression when delivered to cells
the subject of intense interest in understanding and
(63–66). For example, miR-223 is relatively abundant
treating CVD. Treatment strategies currently focus
in plasma, and may transduce an endocrine signal
on systemic anti-miR delivery, which raises concerns
between blood cells and vascular cells (67). Because
for off-target effects, including platelet activation
absolute levels of circulating miRNAs are low, it re-
(79). Future efforts should be aimed at evaluating
mains to be proven whether miRNA transfer is suffi-
cell-type–specific strategies or local delivery. For
cient to
the
achieve
effective
target
repression
in
cardiovascular
system,
enhanced
targeting
could be achieved through the use of adeno-
recipient cells.
Several cardiac miRNAs are detectable in blood
associated
virus
vectors
for
cell-type–specific
early after MI, potentially reducing time to diagnosis
miRNA delivery or nanoparticle-bound anti-miRs or
(68).
miRNA mimics (80).
However,
head-to-head
comparisons
with
established biomarkers, such as high-sensitivity tro-
Preclinical research has focused mainly on identi-
ponins, found that detection of miRNAs did not
fying mechanisms within a single tissue or cell type.
improve on the accuracy or usefulness of current
However, caution needs to be exercised to avoid
methods (69). Furthermore, current miRNA detection
moving
techniques are time consuming and do not allow for
MiRNA regulation of protein expression is highly
the rapid diagnosis required in patients with MI. In
dependent on context and cell type, and their ubiq-
the context of hypertrophic cardiomyopathy, higher
uitous expression makes side effects of miRNA ther-
levels of miR-29a correlate with both hypertrophy
apies unpredictable. Targeting individual miRNAs
and fibrosis (70), but its clinical benefits beside cur-
therefore requires meticulous evaluation of systemic
rent diagnostic tools remain unclear.
effects. A careful approach in advancing miRNA
New biomarker searches should focus on unmet
toward
clinical evaluation
too
quickly.
therapies may slow progression toward clinical
well-
application, but may spare miRNA therapeutics a
performing, established markers already exist. For
setback similar to gene therapy. The great potential of
example, current risk prediction models for MI could
miRNAs justifies the exercise of apprehension before
improve. Three studies, albeit with differing meth-
large-scale clinical studies for CVD.
clinical
needs,
rather
than
areas
where
odologies, have detected differentially expressed
Circulating miRNAs are expressed differentially
miRNAs in patients who went on to suffer acute MI
across disease phenotypes and are implicated as
(71–73). Karakas et al. (71) found a surprisingly strong
novel biomarkers. Their platelet origin could make
correlation of single miRNAs with the risk of cardio-
circulating miRNAs particularly relevant in the
vascular death, although this was in a highly selected
context of CVD. Platelet reactivity may confer car-
population, and was not compared with traditional
diovascular risk, but there is no single accepted
risk models. No single miRNA conferred a clinically
biomarker. More mechanistic studies and validation
significant change in risk of acute MI in either the
in larger cohorts are required to establish the clinical
study by Bye et al. (72) or by Zampetaki et al. (73), but
utility of miRNA biomarkers.
the
combined
usefulness
of
an
miRNA
panel
improved the predictive power of traditional Fra-
REPRINT
REQUESTS
mingham risk models. The miRNAs selected by
Professor
Manuel
AND
Zampetaki et al. (73) also predicted mortality in a
Foundation Centre, King’s College London, 125
cohort of patients with symptomatic coronary artery
Coldharbour Lane, London SE59NU, United Kingdom.
disease (74). Mechanistic links have been reported
E-mail: [email protected].
Mayr,
CORRESPONDENCE:
King’s
British
Heart
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DECEMBER 13, 2016:2577–84
MicroRNAs in Cardiovascular Disease
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KEY WORDS biomarkers, genetic therapy,
noncoding RNA, RNA therapeutic